The role of PKCγ subunit of rACC neurons in the development of bone cancer pain in rats

Background: To explore the role of PKCγ subunit of rACC neurons in the development of bone cancer pain in rats. Methods: 32 adult female SD rats were divided into four groups: blank control group (Naive group), sham operation group (Sham group), bone cancer pain group (BCP group) and PKCγ/shRNA recombinant lentiviral vector group (PKCγ group). 10μl MADB-106 rat mammary cancer cells suspension, 10μl saline was inoculated into the proximal tibia bone marrow cavity of rats in BCP group and Sham group respectively. In the PKCγ group, the rats were taken the same treatment as the BCP group, and then 10μl shRNA/NR2B recombinant lentivirus was injected into the bilateral rACC on the 7th day. The mechanical withdrawal threshold and thermal withdrawal latency were measured every 3 days to assess the rat pain behavior. Immunohistochemistry and Western blotting technology were used to detect the expression of PKCγ subunits in rat rACC neurons after operation. Results: There was no signicant difference in the mechanical withdrawal threshold and thermal withdrawal latency between the four groups (P>0.05). From the 3rd day after operation, the mechanical withdrawal thresholds in BCP group and PKCγ group were signicantly decreased than those in Naive group and Sham group (P<0.05). Compared with the BCP group, the mechanical withdrawal threshold in the PKCγ group increased signicantly (P<0.05). On the 3rd postoperative day, the thermal withdrawal latency in BCP and PKCγ groups was signicantly longer than those in Naive and Sham groups(cid:0)P(cid:0)0.05). From the 10th postoperative day, the thermal withdrawal latency in PKCγ group was shorter than that in BCP group (P<0.05). Western blot analysis showed that the expression of PKCγ in rACC neurons on the 14th day after operation in rats of BCP group was signicantly higher than that in Naive group and Sham group. (P<0.05) However, in the PKCγ group, the expression of PKCγ in rACC neurons was signicantly lower than that in BCP group (P<0.05). Conclusion: Up-regulation of PKC subunit of rACC neurons in bone cancer pain rats may be involved in the development of pain sensitivity in bone cancer.


Background
The pathogenesis of bone cancer pain remains largely unknown, and there is still no effective treatment [1,2].The anterior cingulate cortex (ACC) is an important part of the cerebral cortex, especially the rostral ACC rACC which is associated with pain perception and regulation [3][4][5].
Under the persistent action of noxious stimulation, the function and structure of neurons or synapses in the ACC undergo long-term changes, which is known as plasticity. As an important signaling molecule in cells, PKC plays an important role in neuronal proliferation, differentiation, synapse formation, transmitter release, and long-term potentiation (LTP) of neuronal excitability [6,7]. Previous studies have suggested that PKCγ is involved in the processing of peripheral pain signals and plays an important role in the treatment of noxious stimulation in the dorsal horn of the spinal cord. Does rACC neuronal PKCγ play an important role in the formation of bone cancer pain? In this study, the role of PKCγ subunit of rACC neurons in the development of bone cancer pain was studied in a rat model of bone cancer pain.

Methods
Female adult SD rats, weighing 180-200 g, were provided by the Experimental Animal Center of Shandong University (Jinan, China) All animal procedures were carried out in accordance with the recommendation of the Principles of Laboratory[8] and the ethics committee of the International Association for the Study of Pain [9]. The study was approved by the ethics committee for Animal Care and Use Committees of the Experimental Animal Center of the Second Hospital of Shandong University (Jinan, China) prior to the onset of the experiments (Permit number: KYLL-2017(LW)017. The number of animals was used as little as possible and their suffering was minimized to the lowest degree according to IASP guidelines [9].. All rats were maintained in the following identical conditions: A controlled temperature of 22of˚C, a 12 h light/dark cycle and ad libitum access to food and water. 1 week later, the rats were randomly divided into four groups: normal control group (Naive group), sham operation group (Sham group), Bone cancer pain model group (BCP group) and PKCγ/shRNA recombinant lentiviral vector group (PKCγ group). Normal control group (Naive group): The experimenters were blinded to the pharmacological treatment while processing data and making exclusion decisions. healthy rats without any treatment. Sham operation group (Sham group):unilateral intra-tibial injection of Normal saline. Bone cancer pain model group (BCP group): unilateral intra-tibial injection with 3 μl MADB-106 cells (cell density 5×10 6 /ml) (from Cancer Institute of Concord Medical University of Chinese Academy of Medical Sciences). The initial treatment of PKCγ group was the same as that of BCP group and then rACC was injected bilaterally with shRNA/NR2B recombinant lentivirus 6.2×10 6 particles on the 7th day after operation.
Rat Bone Cancer Pain Model was established as previously described [4]. The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (300m/kg). Super cial incisions were made in the skin overlying the patella to expose the tibia head with minimal damage.
A 23-gauge needle was inserted at the site of intercondylar eminence and pierced 5-10mm below the knee joint into the medullary cavity of tibia. The Needle was then removed and 10 μL of mammary carcinoma cell suspension or 10 μL of heat-killed mammary carcinoma cell suspension into a 50 μL micropipette.
The needle was slowly inserted into the tibia cavity and injected in the carcinoma cells. The injection site was closed with bone wax quickly after the syringe was removed to prevent the cell suspension from leaking out. The wound was sutured to avoid leaving a dead space and was disinfected with iodophors to prevent infection.
Before baseline testing, the rats were habituated to the testing environment for 5 days. Baseline data were tested both before and after using the von Frey hair stimulation. Animals that showed obviously different data between these two tests were discarded. For the remaining animals internalized in the subsequent studies, the average of these two baseline tests was recorded as a baseline data. The experimental rats were placed in a plastic cage (10x10x15cm) with a Plantar Von FreyTM Dynamic Plantar Stimulator at the bottom, and the cage was placed on a wire mesh plate for experimental operation and observation.
After 15 min accommodation, mechanical allodynia was measured as the hind paw withdrawal response to von Frey hair stimulation according to the up-down method. An ascending series of von Frey hair with logarithmically incremental stiffness (1.0, 2.0, 4.0, 6.0, 8.0,15.0 and 20.0g) were applied perpendicularly to the mid-plantar surface (avoiding the less sensitive tori) of each hind paw. The stimulus lasted for 10 seconds, and the interval between each measurement was 10 minutes. The minimum stimulus that caused rat paw withdrawal was de ned as the mechanical withdrawal threshold.
Rats were placed under a cage on a glass plate that was elevated to allow maneuvering of a radiant heat source from below. Controlled radiant heat stimuli were applied to the plantar surface of the hind-paw (BME-410A bolometer, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences). The time from onset of radiant heat application to withdrawal of the rat's hind paw was de ned as the paw withdrawal latency (PWL). The glass plate was kept dry and clean during the measurement. Both hind paws were tested independently with a 5 min interval between trials so that pain can be restored to normal. A cut-off time of 20 s was imposed on the stimulus duration to prevent tissue damage. Each rat was tested on the paw three times and the average value was taken.
A total of 14 days after operation, each group of rats were anaesthetized with pentobarbital prior to the perfusion of 100 ml NS through the ascending aorta and then rapidly sacri ced by decapitation. The rACC tissues were immediately removed (4 rats randomly selected at each time point for measurement) and snap frozen in liquid nitrogen until all the samples were collected for immunohistochemical DAB staining to detect the expression of PKCγ in rACC tissues.On the 7th and 14th day after operation, 4 rats were selected to take rACC brain tissue and the expression level of PKCγ subunit of neurons was assessed by Western blot.
All results from the data analysis are shown as mean ± standard deviation (SD). An ANOVA test, followed by Student-Newman-Keuls (SNK) was used to compare the quantitative data between groups. A p value of less than 0.05 (two-tailed) was considered to indicate a statistically signi cant difference. Graph displays were performed using GraphPad Prism Software version 5.0.

Results
Inter-group comparison of mechanical withdrawal thresholds within the surgical side. There was no statistically signi cant difference between the Naive and Sham groups on the 3rd, 7th, 14th, and 21th days after surgery and preoperatively (P>0.05). However, the BCP and PKCγ groups began to decrease from the 7th day postoperatively and were signi cantly lower than that before surgery. The difference was statistically signi cant. (P<0.05). This trend continued until the 21th day after surgery. From the 7th day of operation, BCP and PKCγ groups were lower than that of Naive and Sham groups. (P<0.05) On the 14th and 21st days after operation, the PKCγ group was signi cantly higher than that of the BCP group (P<0.05) ( Table 1).
Inter-group comparison of mechanical withdrawal duration within the surgical side. There was no statistically signi cant difference between the Naive and Sham groups on the 3rd, 7th, 14th, and 21th days after surgery and preoperatively (P>0.05). Compared with preoperative levels, the BCP and PKCγ groups showed signi cantly prolonged mechanical withdrawal duration from 7 days after surgery (P<0.05 or P<0.01). This difference continued until the 21th day postoperatively. From the 7th day postoperatively, the mechanical withdrawal duration of the BCP and PKCγ groups was higher than that of the Naive and Sham groups (P<0.05 or P<0.01). On the 14th and 21st days after operation, the PKCγ group was signi cantly lower than that of the BCP group (P<0.05 or P<0.01) ( Table 2) Expression of PKCγ protein in rACC neurons of rats in each group on the 7th day after operation (without recombinant PKCγ/shRNA in rACC). In order to assess PKCγ protein expression in rACC neurons following the development of mechanical pain and thermal sensitization in rats rats' rACC brain tissue was taken on the 7th day after intrapatellar injection of breast cancer cells when the rats showed mechanical pain and thermal pain. The expression of PKCγ in the brain of rats in each group was exmined by western blot. The expression levels of PKCγ protein in the BCP and PKCγ groups were higher than those in the Naive and Sham groups, (P<0.05) (Fig 1).
The expression of PKCγ protein in rACC neurons of rats in each group on the 14th postoperative day (rACC-administered recombinant lentiviral vector of PKCγ/shRNA). In order to examine the changes of PKCγ protein expression in rACC neurons, rACC brain neurons were taken from each group after rACC neurons were given with PKCγ/shRNA recombinant lentivirus vector. Compared with the Naive and Sham groups, the expression of PKCγ protein was up-regulated in the BCP and PKCγ groups (P<0.05) as assessed by Western blotting (P<0.05). Compared with the BCP group, the expression of PKCγ protein in the PKCγ group was decreased (P<0.05) (Fig 2).
Comparison of the number of bilateral PKCγ-positive neurons in rACC after rACC-administered recombinant lentiviral vector of PKCγ/shRNA by immunohistochemistry. PKCγ-positive neurons in rACC neurons were assessed after PKCγ/shRNA recombinant lentiviral vector administration in rACC of rats in each group on the 14th day after operation. We observed an increased expression of PKCγ protein in the BCP and PKCγ groups compared with the Naive and Sham groups (P<0.05). However, compared with the BCP group, the expression was decreased in the PKCγ group (P<0.05) (Fig 3).

Discussion
This study found that changes in pain-related behaviors such as mechanical withdrawal threshold and mechanical withdrawal duration in rats with bone cancer pain resulted in up-regulation of PKCγ subunit expression in rACC neurons. Recombinant lentiviral vector of PKCγ/shRNA was administered in rACC to silence the PKCγ subunit, resulting in reduced pain in rats. These results indicate that the PKC subunit of rACC neurons plays an important role in the development of pain sensitivity in bone cancer.
Pain is one of the symptoms in terminal cancer patients and has been described as a deep, burning-like pain with severe emotional reactions. So far, although there are opioid, diphosphonate, radiotherapy, chemotherapy and surgery for relieving cancer pain, it is still reported that many cancer patients have inadequate and undermanaged pain control [10,11], One of the reasons may be related to the unclear mechanism of bone cancer pain. Therefore, it is of important clinical signi cance to explore the mechanism of bone cancer pain for improving the treatment of patients. Recent studies have shown that the excitability of neurons on the upper spinal cord such as ACC and the enhancement of synaptic transmission play an important role in the development of chronic pain [12]. The ACC is an important structure of the limbic system and re ect affective and motivational aspects of pain.
Many study results showed that ACC especially rostral ACC participated in nocuity information transmission and regulation. Unpleasant sensations caused by noxious stimuli or pain can also activate ACC [13]. Resection of peripheral cortical tissue including ACC can reduce the patient's pain and emotional response, but not affect the intensity and location of the pain stimulus. The latest BOLD further directly proves the important role of ACC in emotional regulation in human subjects. When given a certain stimulus, the negative emotions increase and the activity of ACC also rapidly increases [14][15][16]. These studies suggest that enhanced nerve excitability of rACC plays an important role in the generation of bone cancer pain. PKCγ as an important signal transduction molecule in neurons activates the protein kinase system in neurons, thereby changes the phosphorylation state of the substrate. PKCγ is also involved in synaptic remodeling of neurons and long-term potentiation. Studies have shown PKCγ in peripheral nerve and spinal dorsal horn plays an important role in the processing of noxious stimulation which suggests that PKCγ in rACC neurons may be vital in the enhancement of nerve excitability and pain sensitivity of rACC neurons, and no relevant reports have been reported. The results of this study showed that up-regulation of PKCγ subunit expression in rACC neurons was accompanied by pain in rats, suggesting that this subunit may be involved in the development of pain sensitivity in bone cancer. To further explore whether the PKC subunit of rACC neurons play a major role in bone cancer pain, we injected PKCγ/shRNA recombinant lentiviral vectors into the bilateral rACC to silence the PKCγ subunits of rACC neurons. We observed an increase of the mechanical withdrawal threshold and the shortening of the mechanical withdrawal duration, suggesting that rat's hyperalgesia was reduced. This con rms that the PKCγ subunit of rACC neurons plays an important role in the development of rat bone cancer pain.
Malmberg found that acute pain in PKCγ knockout mice was not affected signi cantly, while chronic pain was weakened which is consistent with the present study [17]. Follow-up study should exclude more in uencing factors and get more accurate results.

Conclusion
In summary, this study con rmed that up-regulated expression of PKCγ subunit of rACC neurons in bone cancer pain rats was involved in the development of pain sensitivity in bone cancer.

Declarations
Ethics approval and consent to participate All experimental procedures and animal handling were performed according to both the Guiding Principles for the Care and Use of Laboratory Animals. The protocol was approved by the committee on the Ethics of Animal Experiments of the Shandong University.

Consent for publication
Not applicable Availability of data and material The datasets generated and analysed during the current study are not publicly available due to copyright issues, but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.    Western blotting in the expression of PKCγ subunits in neurons of ACC brain regions in rats 7 days after operation.*P < 0.05 compared with naïve group; #P <0.05, compared with Sham group.